Electrochemical detection of 17beta-estradiol using DNA aptamer immobilized gold electrode chip

An electrochemical detection method for chemical sensing has been developed using a DNA aptamer immobilized gold electrode chip. DNA aptamers specifically binding to 17beta-estradiol were selected by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) process from a random ssDNA library, composed of approximately 7.2 x 10(14) DNA molecules. Gold electrode chips were employed to evaluate the electrochemical signals generated from interactions between the aptamers and the target molecules. The DNA aptamer immobilization on the gold electrode was based on the avidin-biotin interaction. The cyclic voltametry (CV) and square wave voltametry (SWV) values were measured to evaluate the chemical binding to aptamer. When 17beta-estradiol interacted with the DNA aptamer, the current decreased due to the interference of bound 17beta-estradiol with the electron flow produced by a redox reaction between ferrocyanide and ferricyanide. In the negative control experiments, the current decreased only mildly due to the presence of other chemicals.     

Construction of novel single-cell screening system using a yeast cell chip for nano-sized modified-protein-displaying libraries

A novel screening system using a microchamber array chip was developed for construction of combinatorial nano-sized protein libraries in combination with yeast cell surface engineering. It is possible to place a single yeast cell into each microchamber, to observe its behavior, and to pick up the target cell. The microchamber array chip is referred to as a “yeast cell chip.” A single EGFP-displaying yeast cell could be detected, picked up by a micro-manipulator, and cultivated on agar medium. Furthermore, a catalytic reaction, the hydrolysis of fluorescein dioctanate, by a single yeast cell displaying Rhizopus oryzae lipase (ROL) was carried out in one microchamber. The ROL-encoding gene in a single ROL-displaying cell was amplified by PCR. These results demonstrate that this yeast cell chip in combination with cell surface engineering could be used as a tool in a high-throughput screening system not only for a single living cell and a whole-cell catalyst with a nano-sized protein cluster but also for modified nano-sized and functional protein molecules from protein libraries on the cell surface.     

A microbial chip combined with scanning electrochemical microscopy.

A microbial chip for bioassay was fabricated and its performance was characterized by scanning electrochemical microscopy (SECM). The microbial chip was prepared by spotting a suspension of Escherichia coli on a polystyrene substrate by using a glass capillary pen. The respiration activity of the E. coli spot was imaged with SECM by mapping the oxygen concentration around the spot. The SECM images of the microbial chips clearly showed spots with lower reduction currents, indicating that E. coli in the spots uptake oxygen by respiration. The bactericidal effects of antibiotics (streptomycin and ampicillin) were measured using the E. coli-based microbial chip, and discussed in comparison with the minimum inhibitory concentration (MIC) determined by an agar plate dilution method.     

Molecular screening of free-living microbial eukaryotes: diversity and distribution using a meta-analysis

Numerous environmental gene library studies have shown that eukaryote microbial diversity is much greater than expected. Molecular surveys of several 'extreme' and some more anthropomorphically commonplace environments have revealed many previously unsampled micro-eukaryotic lineages. However, it cannot be assumed that all of the sequences recovered from these studies are derived from real organisms, and for those that are, many questions remain about their distribution and ecology. Integrating all available sequence data from these studies reveals patterns of distribution, diversity and evolutionary relationships that are not accessible from independent analyses of the individual surveys and enables us to review the wider implications of such studies.     

Electrochemical changes in media due to microbial

Microbial metabolism affected the electrical impedance parameters of a two terminal-measuring cell-containing growth media. The relationship between microbial growth and relative changes in both The capacitive and resistive parts of impedance was examined. Both components of impedance were shown to be indicative of bacterial growth. In low conductivity media the change in the conductance of the media (G_sol) clearly correlated to bacterial growth. In more conductive media the relative changes in G_sol were smaller, and in these media measurements of the changes of polarization capacitance (C_pol) were useful for monitoring bacterial growth.Yeast growth in two media resulted in large changes in C_pol (20–100%) while the changes in G_sol were very small (1–4%). This result indicated that, for some combinations of microorganisms and media, measuring C_pol might be preferable over G_sol for the detection of microbial growth.Microbial metabolism resulted in a change of 2–2.5 units in pH. This pH change resulted in a 40% change in C_pol but less than a 14% change in G_sol.     

用于药物筛选的微流控细胞阵列芯片

细胞区域分布培养以及如何有效地对微流体进行操控是微流控阵列芯片在细胞药物研究中的关键技术。本研究介绍了一种利用SU-8负性光刻胶模具和PDMS制作双层结构的微流控细胞阵列芯片的方法,该芯片通过C型的坝结构将进样细胞拦截在芯片的细胞培养的固定区域,键合双层PDMS构成阀控制层,阀网络的开关作用成功实现了芯片通道内微流体的操控,同时芯片设计了药物浓度梯度网络,产生6个不同浓度的药物刺激细胞。通过对芯片3种共培养细胞活性的检测和药物伊立替康(CTP-11)对肝癌细胞的浓度梯度刺激等实验结果验证该芯片在细胞研究和药物筛选等方面的可行性。     

A reverse genetic screen in Drosophila using a deletion-inducing mutagen

We report the use of the cross-linking drug hexamethylphosphoramide (HMPA), which introduces small deletions, as a mutagen suitable for reverse genetics in the model organism Drosophila melanogaster. A compatible mutation-detection method based on resolution of PCR fragment-length polymorphisms on standard DNA sequencers is implemented. As the spectrum of HMPA-induced mutations is similar in a variety of organisms, it should be possible to transfer this mutagenesis and detection procedure to other model systems.     

Single-species microbial biofilm screening for industrial applications

While natural microbial biofilms often consist of multiple species, single-species biofilms are of great interest to biotechnology. The current study evaluates biofilm formation for common industrial and laboratory microorganisms. A total of 68 species of biosafety level one bacteria and yeasts from over 40 different genera and five phyla were screened by growing them in microtiter plates and estimating attached biomass by crystal violet staining. Most organisms showed biofilm formation on surfaces of polystyrene within 24 h. By changing a few simple conditions such as substratum characteristics, inoculum and nutrient availability, 66 strains (97%) demonstrated biofilm formation under at least one of the experimental conditions and over half of these strains were classified as strong biofilm formers, potentially suitable as catalysts in biofilm applications. Many non-motile bacteria were also strong biofilm formers. Biofilm morphologies were visualized for selected strains. A model organism, Zymomonas mobilis, easily established itself as a biofilm on various reactor packing materials, including stainless steel.     

SPR蛋白质芯片在输入性疟疾筛查中的应用

输入性疟疾已是我国疟疾防控的主要危险因素,如何对入境人员进行疟疾快速筛查是急需解决的难题。蛋白质芯片已被广泛应用于高通量筛选和诊断,本研究尝试构建了表面等离子共振技术 (Surface plasmon resonance,SPR) 蛋白芯片用于恶性疟疾的快速检测。采用聚乙二醇高分子处理的特异性吸附表面,以恶性疟疾特异性抗原富组氨酸蛋白Ⅱ (Histidine-rich protein Ⅱ,HRP2) 作为捕获探针,建立疟疾的微阵列芯片,并对芯片的最佳抗原固定浓度,检测的灵敏性和特异性,以及抗干扰能力进行了分析。该芯片可成功应用于恶性疟疾的筛查,具有无标记、即时快速的特点,与荧光定量PCR法相比,两种方法在敏感度和特异性方面无统计学差异。研究结果为一步研制疟疾分型鉴定蛋白质芯片奠定了基础,有利于对入境人员进行疟疾快速筛查。     

Metabolism of the mutagen MeIQx in vivo: metabolite screening by liquid chromatography-thermospray mass spectrometry

2-Amino-3,8-dimethylimidazo(4,5-f)quinoxaline (MeIQx) is a potent mutagen found in cooked food. MeIQx and its isotopically labelled (13C, 15N2 and 14C) analogues were synthesised and used for metabolic studies in vivo. An equimolar mixture of MeIQx and its 13C, 15N2 stable isotope labelled analogue (containing tracer amounts of 14C-MeIQx) was given intraperitoneally to mice. Some 67% of the radioactivity was eliminated in urine and faeces within 24h. Four radiolabelled species were observed when urine was analysed by HPLC, corresponding to unchanged MeIQx and three more polar metabolites. Urine was analysed directly by HPLC-thermospray mass spectrometry. Four signals were observed containing the characteristic 1:1 isotopic doublet, corresponding to unchanged MeIQx, an MeIQx glucuronide, and two uncharacterized metabolites.     

Electrochemical screening of recombinant protein solubility in Escherichia coli using scanning electrochemical microscopy (SECM)

A microbial array chip with collagen gel spots entrapping living Escherichia coli (E. coli) DH5alpha was applied for the screening of recombinant protein solubilities. The alpha-fragment of beta-galactosidase (betaGal) was fused to the target protein, namely, maltose-binding protein (MBP), to monitor the solubility of MBP. Scanning electrochemical microscopy (SECM) was used to detect the release of p-aminophenol from E. coli cells catalyzed by intracellular betaGal. Comparison of the SECM-based method with the Western blotting-based method indicated that the current response obtained using SECM increased with an increase in the betaGal activity and therefore, with the soluble fraction of MBP in the host cells.     

Amperometric detection of the bacterial metabolic regulation with a microbial array chip

A microbial array chip with collagen gel spots entrapping living bacterial cells has been applied to investigate the metabolic regulation in Paracoccus denitrificans. Scanning electrochemical microscopy (SECM) was used to monitor the ferrocyanide production that reflects the electron flow in the respiratory chain located within the internal membrane of P. denitrificans. The ferrocyanide production from P. denitrificans largely depends on the types of the carbon source (glucose or lactate), suggesting that the electron flow rate in the respiratory chain depends on the activity of the metabolic pathway located up-stream of the respiratory chain. More importantly, it was found that the enzymes affecting glucose catabolic reactions were significantly up-regulated in cultures with a nutrient agar medium containing D-(+)-glucose as a sole carbon source. Enzyme assays using crude extracts of P. denitrificans were carried out to identify the enzymes expressed at a higher level in cultures supplemented with D-(+)-glucose. It was confirmed that the pyruvate kinase and enzymes of the overall Entner-Doudoroff pathway were highly induced in cultures containing D-(+)-glucose.     

A new screening method for investigating microbial interactions

Interactions among Candida albicans, Staphylococcus aureus and Escherichia coli were investigated using a screening system in which test micro-organisms were incorporated in agar discs and effector micro-organisms in fluid growth media. Total as well as partial inhibition of test micro-organisms was observed in agar discs when these were incubated in broths containing effector micro-organisms. The ratio of numbers of test to effector micro-organisms was found to be of importance in the inhibition effect. The technique was found to be cheap, simple and versatile.     

High-throughput system-wide engineering and screening for microbial biotechnology

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